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The Calvin-Benson-Bassham cycle (CBBC) performs carbon fixation in photosynthetic organisms. Among the eleven enzymes that participate in the pathway, sedoheptulose-1,7-bisphosphatase (SBPase) is expressed in photo-autotrophs and contributes to the regeneration of ribulose-1,5-bisphosphate, the carbon fixation co-substrate used by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While SBPase is structurally similar to fructose-1,6-bisphosphatase (FBPase) involved in both neoglucogenesis and the CBBC, it exclusively functions in the CBBC and is indispensable for building a productive cycle. In this study we report the first structure of an SBPase from a chlorophyte, the model unicellular green microalgaChlamydomonas reinhardtii. By combining experimental and computational structural analyses, we describe the topology, conformations and quaternary structure ofChlamydomonas reinhardtiiSBPase. We identify active site residues and locate sites of redox- and phospho-post-translational modifications that contribute to enzymatic functions. Finally, we observe that CrSBPase adopts distinct oligomeric states that may dynamically contribute to the control of its activity.

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an α8β8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs., In the No-Go decay mRNA surveillance pathway, mRNAs containing stalled ribosomes are cleaved by an endoribonuclease. Here the authors show the endonucleolytic cleavage on the artificial No-Go decay target mRNAs, revealing downstream decay process by Trl1 kinase and the 5′ to 3′ exonuclease Dxo1.

International audience; Residue coevolution within and between proteins is used as a marker of physical interaction and/or residue functional cooperation. Pairs or groups of coevolving residues are extracted from multiple sequence alignments based on a variety of computational approaches. However, coevolution signals emerging in subsets of sequences might be lost if the full alignment is considered. iBIS2Analyzer is a web server dedicated to a phylogeny-driven coevolution analysis of protein families with different evolutionary pressure. It is based on the iterative version, iBIS2, of the coevolution analysis method BIS, Blocks in Sequences. iBIS2 is designed to iteratively select and analyse subtrees in phylogenetic trees, possibly large and comprising thousands of sequences. With iBIS2Analyzer, openly accessible at http://ibis2analyzer.lcqb.upmc.fr/, the user visualizes, compares and inspects clusters of coevolving residues by mapping them onto sequences, alignments or structures of choice, greatly simplifying downstream analysis steps. A rich and interactive graphic interface facilitates the biological interpretation of the results.

Machine Learning techniques, Neural Networks in particular,are going through an impressive expansion, permeating various domains, becoming the next frontier forhuman societies. Autonomous vehicles, aircraft collisionavoidance, cancer detection, justice advisors, or mooring line failure detection are but a few examples of Neural Networks applications.This effervescence, however, may hold more than benefits,as it slowly but surely reaches critical systems.Indeed, the remarkable efficiency of neural nets comes at a price, more and more underlined by the scientific consensus : weakness to environmental or adversarial perturbations, unpredictability... which prevents their full-scale integration into critical systems. While the domain of critical software enjoys a plethora of methods that help verify and validate software (abstract interpretation, model checking, simulation, bounded tests...),these methods are generally useless when it comes to Neural Nets.This thesis aims at bridging formal software verification and machine learning, in order to bring trust in critical systems incorporating Neural Networks elements.; L'apprentissage machine, en particulier au moyen des réseaux de neurones artificiels, connaît depuis une dizaine d'année une expansion impressionnante.Véhicules autonomes, capteurs d'anomalies d'ancrage sur des plateformes offshores, détecteurs de collision d'aéronefs,aide au diagnostic pour différents cancers sont autant d'applications faisant intervenir les technologies d'apprentissage profond au sein de systèmes critiques;ouvrant des perspectives inexplorées pour les sociétés humaines. Bien que bénéfique en apparence,cette révolution a de quoi inquiéter à mesure qu'elle se concrétise : la fragilité de ces techniques d'apprentissage est désormais un fait scientifique établi.La taxonomie des vulnérabilités, qu'elles soient accidentelles ou malicieuses, remet en question la possibilité d'intégrer des réseaux de neurones dans des domaines critiques qui pourraient pourtant en bénéficier. A l'heure actuelle, peu de méthodes permettent de démontrer formellement la fiabilité d'un réseau de neurones.Par contraste, le domaine du logiciel critique, quant à lui, jouit d’une multitude de méthodes et techniques : vérification formelle, model checking, simulation, interprétation abstraite, tests dirigés, etc. L'objectif de cette thèse est de réconcilier l'abondance des techniques de vérification de programmes classiques et l'absence de garanties sur les réseaux de neurones, ce pour permettre aux logiciels critiques de conserver le haut niveau de confiance qu'ils ont atteint quand ils seront inévitablement modifiés avec des mécanismes d'apprentissage machine.

International audience; Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin–Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX–target recognitions.

National audience; La grande polyvalence et les résultats impressionnants des réseaux de neurones modernes viennent en partie de leur non-linéarité. Cette propriété fondamentale rend malheureusement très difficile leur vérification formelle, et ce, même si on se restreint à une structure linéaire par morceaux. Cependant, chacune de ces régions linéaires prise indépendamment est simple à analyser. Nous proposons dans cet article une méthode permettant de simplifier le problème de vérification en opérant une séparation en multiples sous-problèmes linéaires. Nous présentons également des résultats concernant la structure de ces régions linéaires ainsi que leur similarité. Ce travail en cours démontre déjà la faisabilité de l'approche sur des problèmes simples ainsi que quelques expériences face à l'état de l'art.

Significance Defects in RNA quality control pathways manifest as disease because they function to selectively remove aberrant or defective species to ensure cellular homeostasis. The RNA exosome and TRAMP complexes encompass key components of the RNA surveillance machinery. Here we show that exoribonuclease activities of Rrp6-associated RNA exosomes protect stable RNA species from TRAMP-mediated polyadenylation and degradation, and that each catalytic activity of the RNA exosome-TRAMP complex contributes to substrate discrimination and degradation of less stable RNA species. Our results highlight a previously unappreciated role for Rrp6 in protecting stable RNA species from degradation and suggest additional mechanistic analogies between RNA and protein quality control pathways with respect to proofreading mechanisms that protect stable species while targeting unstable species for destruction., Quality control requires discrimination between functional and aberrant species to selectively target aberrant substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation followed by helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry, we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to protect stable RNA from degradation while selectively targeting and degrading less stable RNA. Substrate discrimination is lost when the distributive exoribonuclease activity of Rrp6 is inactivated, leading to degradation of stable and unstable RNA species. These data support a proofreading mechanism in which deadenylation by Rrp6 competes with Mtr4-dependent degradation to protect stable RNA while selectively targeting and degrading unstable RNA.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant thioredoxins evolved into seven types with diverse subcellular localization and distinct protein targets selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the first crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows TRXz with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. TRXz distinct electronegative surface thereby extends the repertoire of TRX-target recognitions.

There is an urgent need for specific antiviral drugs directed against SARS-CoV-2 both to prevent the most severe forms of COVID-19 and to reduce viral excretion and subsequent virus dissemination; in the present pandemic context, drug repurposing is a priority. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus in order to inhibit its association with viral RNA could be a strategy to impeding viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, belonging to the NSAID family, previously demonstrated against Influenza A virus, were evaluated against SARS-CoV-2. Naproxen binding to the nucleoprotein of SARS-CoV2 was shown by molecular modeling. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2 induced-damage. The benefit of naproxen addition to the standard of care is tested in an on-going clinical study.

The Calvin&ndash, Benson cycle is the key metabolic pathway of photosynthesis responsible for carbon fixation and relies on eleven conserved enzymes. Ribose-5-phosphate isomerase (RPI) isomerizes ribose-5-phosphate into ribulose-5-phosphate and contributes to the regeneration of the Rubisco substrate. Plant RPI is the target of diverse post-translational modifications including phosphorylation and thiol-based modifications to presumably adjust its activity to the photosynthetic electron flow. Here, we describe the first experimental structure of a photosynthetic RPI at 1.4 &Aring, resolution. Our structure confirms the composition of the catalytic pocket of the enzyme. We describe the homo-dimeric state of the protein that we observed in the crystal and in solution. We also map the positions of previously reported post-translational modifications and propose mechanisms by which they may impact the catalytic parameters. The structural data will inform the biochemical modeling of photosynthesis.

RESUME L’arret cardiaque extra-hospitalier represente un probleme de sante publique majeur, avec plus de 40 000 evenements chaque annee en France. Moins de 10 % des patients survivent. Les premieres minutes de prise en charge sont cruciales, et une reanimation adaptee initiale plus systematique par le temoin, associant massage cardiaque externe et usage d’un defibrillateur, devrait permettre d’atteindre des taux de survie plus eleves. Un programme ambitieux et coordonne base sur l’education de toute la population aux gestes qui sauvent, des le jeune âge, est indispensable pour ameliorer le pronostic. L’Academie nationale de medecine propose dans ce document une strategie, identifiant quatre mesures essentielles pour une meilleure education de la population aux gestes qui sauvent.